DNA transposons are mobile genetic elements that can change positions within the genome(s) utilizing a “cut-and-paste” mechanism, and have an important value in the genetic modification of biological genome. Two essential sequence elements participate in transposition: (1) flanked inverted terminal repeats (ITRs) involved in specific recognition and (2) active transposases. An autonomous DNA transposon could usually transcribe and translate active transposase to catalyze the transposition of the transposon. However, the coding regions for transposase in most of the vertebrate DNA transposons mutated and lost their activity during the evolutionary process because of the vertical inactivation in hosts. Even so, the coding regions for transposase can be reconstructed according to conserved sequence for transposase in inactivated transposon by bioinformatics, such as the artificially constructed Sleeping Beauty transposition system and Frog Prince transposition system that could mediate transposition in animal embryos or cells.
Several autonomous transposons have been successively discovered in fish in the last decade. For example, Tol2 transposon, which is identified in medaka and belongs to hAT (hobo-Ac-Tam3) transposon superfamily, is the first natural active transposon found in vertebrates. The transcribed mRNA in vitro of Tol2 transposase can mediate efficient insertion of transposable elements, and can achieve efficient transposition in the cells of vertebrates including zebrafish, clawed frog, chicken and mammals. Goldfish Tgf2 transposon belonging to hAT transposon superfamily is also a natural active transposon. The Tgf2 transposon system can efficiently transpose in the embryos of goldfish, zebrafish and the other Cyprinoid fishes (Reference 1: X. Y. Jiang, X. D. Du, Y. M. Tian, R. J. Shen, C. F. Sun, S. M. Zou, Goldfish transposase Tgf2 presumably from recent horizontal transfer is active, FASEB J. 26 (2012) 2743-2752. Reference 2: L. D. Cheng, X. Y. Jiang, Y. M. Tian, J. Chen, S. M. Zou, The goldfish hAT-family transposon Tgf2 is capable of autonomous excision in zebrafish embryos, Gene (2013) http://dx.doi.org/10.1016/j.gene.2013.11.084.). Additionally, a Tcl-like transposon from flatfish (Pleuronectes platessa), Passport, also has transposition activity in the vertebrate cells.
Recently, the common method is that the donor plasmid containing the left and right ends of the transposon and transposase mRNA are coinjected into fertilized fish eggs at the 1-2 cell stage. The transposase mRNA is firstly translated to transposase protein after injection into fertilized eggs, which then mediates the insertion of target gene or element into the fish genome. The transcription of the transposase mRNA needs to be completed in vitro, but the general fish farms do not have the conditions for synthesis and cryopreservation. In addition, the translation of transposase takes some time, and usually produces transgenic chimeras (T. Shibano, M. Takeda, I. Suetake, K. Kawakami, M. Asashima, S. Tajima, M. Taira, Recombinant Tol2 transposase with activity in Xenopus embryos, FEBS Lett. 581 (2007) 4333-4336.). However, the occurrence of chimeras can be effectively prevented by the direct injection of active transposase protein.
The reference 1 (X. Y. Jiang, X. D. Du, Y. M. Tian, R. J. Shen, C. F. Sun, S. M. Zou, Goldfish transposase Tgf2 presumably from recent horizontal transfer is active, FASEB J. 26 (2012) 2743-2752.) reported that an active Tgf2 transposon of hAT transposon superfamily was found in goldfish, and it had 7 kinds of mRNA transcripts, which encoded putative transposases of 3 different lengths of 686 (gfTP1), 650 (gfTP2-3), and 577 (gfTP4-7) aa residues. The active recombinant goldfish Tgf2 transposase protein has not been reported at present.
Recombinant protein is the protein obtained by DNA or RNA recombinant technique. The route can be divided into in vitro and in vivo methods, which are all based on gene recombination technology that recombinant vector connected gene fragment that can be translated into target protein is constructed, and then transferred to the host cell that can express target protein to express the particular recombinant protein molecule. There are usually some problems in the recombinant protein expression process concluding that the recombinant protein could not be expressed, the expression level is low, the expression product has no or a low activity, etc. Many factors affect the expression level and activity of the recombinant protein, such as the modification of target gene in the gene level, the selection of promoter, the inducer concentration, induction temperature and induction time in the bacterial fermentation, etc. The recombinant protein production is a process requiring theory and practice, and many problems need to be solved in order to obtain an optimal construction, expression and purification scheme.